Single-molecule imaging reveals multiple pathways for the recruitment of translesion polymerases after DNA damage

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Unrepaired DNA lesions stall DNA replication forks, causing genomic instability and cell death.
• Translesion synthesis (TLS) is a critical DNA damage tolerance pathway that utilizes specialized polymerases to bypass lesions.
• The precise mechanisms by which TLS polymerases access the DNA template at stalled replication forks are not fully understood.

Purpose of the Study:

• To investigate the recruitment dynamics of DNA polymerase IV (Pol IV) to sites of DNA damage in live Escherichia coli cells.
• To elucidate the factors and mechanisms governing Pol IV access to DNA lesions during replication stress.

Main Methods:

• Utilized particle-tracking photoactivated localization microscopy (PALM) to image live Escherichia coli.
• Genetically engineered cells with a functional fusion of endogenous Pol IV to photoactivatable fluorescent protein PAmCherry.
• Observed Pol IV localization in response to DNA damage and varying lesion types.

Main Results:

• Pol IV exhibited significant enrichment at replication sites exclusively upon induction of DNA damage.
• The recruitment mechanism of Pol IV was found to be dependent on the specific type of DNA lesion encountered.
• Identified roles for interactions with proteins beyond the known processivity factor β in Pol IV recruitment under certain conditions.

Conclusions:

• Pol IV recruitment to DNA damage sites is a dynamic process regulated by lesion identity.
• Multiple protein interactions, modulated by the nature of the DNA lesion, mediate Pol IV access for translesion synthesis.
• These findings provide novel insights into the regulation of DNA repair pathways and genomic stability.